Ubiquitin, a 76-residue protein, is present in eukaryotes either free or covalently joined, through its carboxyl-terminal glycine residue, to various cytoplasmic, nuclear, and integral membrane proteins. The coupling of ubiquitin to such proteins is catalyzed by a family of ubiquitin-conjugating enzymes (also called E2 enzymes). The fact that the amino acid sequence of ubiquitin is conserved among eukaryotes to an extent unparalleled among known proteins suggested that ubiquitin mediates a basic cellular function. However, the biological role of ubiquitin remained a mystery until relatively recently.
Ubiquitin has been found to be one of several factors required for ATP-dependent protein degradation in eukaryotic cells. One function of intracellular protein degradation, most of which is ATP-dependent, is selective elimination of damaged and otherwise abnormal proteins. Another is to confer short half-lives on undamaged proteins whose concentrations in the cell must vary as functions of time, as is the case, for example, with many regulatory proteins. Many other proteins, while long-lived as components of larger macromolecular complexes such as ribosomes and oligomeric proteins, are metabolically unstable in a free, unassociated state. (The term "metabolic instability" implies a relatively short half-life of a protein as a physical entity in vivo, with the adjective "metabolic" being used to distinguish this property from "stability" as such, which often means either conformational or chemical stability of a protein, but not its existence as a physical entity in vivo.)
Recent work has shown that selective degradation of many short-lived proteins requires a preliminary step of ubiquitin conjugation to a targeted proteolytic substrate. It was proposed that one role of ubiquitin is to serve as a signal for attack by proteases specific for ubiquitin-protein conjugates (reviewed by Finley and Varshavsky, Trends Biochem. Sci. 10:343-348 (1985)).
This understanding, however, left unsolved the problem of targeting: how are intracellular proteins initially recognized as proteolytic substrates? At least some short-lived proteins are recognized as such because they contain sequences (degradation signals) which make these proteins substrates of specific proteolytic pathways. The first degradation signal to be understood in some detail comprises two distinct determinants: the protein's amino-terminal residue and a specific internal lysine residue (Bachmair et al., Science 234:179-186 (1986); Bachmair and Varshavsky, Cell 56:1013-1032 (1989)). The N-end rule a code that relates the protein's metabolic stability to the identity of its amino-terminal residue (Bachmair et al., Science 234:179-186 (1986)), is universal in that different versions of the N-end rule operate in all of the eukaryotic organisms examined, from yeast to mammals (Gonda et al., J. Biol. Chem. 264:16700-16712 (1989)).
The second essential determinant of the N-end rule-based degradation signal, referred to herein as the second determinant, is a specific internal lysine residue in the substrate protein that serves as the site of attachment of a multiubiquitin chain. Formation of the multiubiquitin chain on a targeted short-lived protein is essential for the protein's subsequent degradation (FIG. 1). The enzymatic conjugation of ubiquitin to other proteins involves formation of an isopeptide bond between the carboxy-terminal glycine residue of ubiquitin and the .epsilon.-amino group of a lysine residue in an acceptor protein. In a multiubiquitin chain, ubiquitin itself serves as an acceptor, with several ubiquitin moieties attached sequentially to an initial acceptor protein to form a chain of branched ubiquitin-ubiquitin conjugates (Chau et al., Science 243:1576-1583 (1989)).
The elucidation of the fundamental rules governing the metabolic stability of proteins in cells, and especially the deciphering of the N-end rule-based degradation signal, has made possible the manipulation of proteins to vary their half-lives in vivo (Bachmair and Varshavsky, Cell 56:1019-1032 (1989)). A more detailed understanding of these degradation signals, their components, and their interrelationships is necessary in order to realize the full potential of this powerful methodology.